Beta-HPV 5 and 8 E6 promote p300 degradation by blocking AKT/p300 association

Abstract

The E6 oncoprotein from high-risk genus alpha human papillomaviruses (α-HPVs), such as HPV 16, has been well characterized with respect to the host-cell proteins it interacts with and corresponding signaling pathways that are disrupted due to these interactions. Less is known regarding the interacting partners of E6 from the genus beta papillomaviruses (β-HPVs); however, it is generally thought that β-HPV E6 proteins do not interact with many of the proteins known to bind to α-HPV E6. Here we identify p300 as a protein that interacts directly with E6 from both α- and β-HPV types. Importantly, this association appears much stronger with β-HPV types 5 and 8-E6 than with α-HPV type 16-E6 or β-HPV type 38-E6. We demonstrate that the enhanced association between 5/8-E6 and p300 leads to p300 degradation in a proteasomal-dependent but E6AP-independent manner. Rather, 5/8-E6 inhibit the association of AKT with p300, an event necessary to ensure p300 stability within the cell. Finally, we demonstrate that the decreased p300 protein levels concomitantly affect downstream signaling events, such as the expression of differentiation markers K1, K10 and Involucrin. Together, these results demonstrate a unique way in which β-HPV E6 proteins are able to affect host-cell signaling in a manner distinct from that of the α-HPVs.

Figure 1. E6 from α and β HPVs bind p300 with different strengths.

(A) Representative immunoblot showing the levels of p300 co-precipitating with various GST-tagged E6 proteins. Equal levels of input GST-E6 are demonstrated by the GST immunoblot in the lower portion of the figure. (B) HFKs expressing E6-HA were immunoprecipitated with a HA antibody (HA) or mouse Immunoglobulin G (IgG) as a negative control and immunoblotted with a p300 and HA antibody. (C) Identical cell lysates were immunoprecipitated with a p300 antibody (p300) or IgG control and immunoblotted with a p300 and HA antibody. Input is equal to 5% of total protein lysate.

Figure 2. β HPV 5 and 8E6 binding to p300 leads to p300 degradation.

(A) Representative immunoblot showing the levels of p300 in LXSN and E6-expressing HFKs. Actin is shown as a loading control. (B) p300 mRNA levels in LXSN and E6-expressing HFKs. Relative levels of p300 mRNA were calculated using the ΔΔCT method with GAPDH to normalize mRNA levels within each sample. Values shown are the mean fold-change in each sample compared to the LXSN vector control. Error bars represent the standard deviation for each sample (n = 3). (C) 5E6 and 8E6 mRNA levels following transfection of E6 siRNA. Relative levels of 5 and 8E6 mRNA were calculated using the ΔΔCT method with GAPDH to normalize mRNA levels within each sample. Values shown are the mean fold-change in each sample for one representative experiment. (D) Representative immunoblot showing the levels of p300 in LXSN, 5E6 and 8E6 expressing cells following transfection with siRNAs specific for each E6. Actin is shown as a loading control. siRNA #1* and #2* in LXSN cells represent a 50/50 mixture of 5E6 siRNA #1 and 8E6 siRNA #1, and 5E6 siRNA #2 and 8E6 siRNA #2 respectively.

Figure 3. β HPV 5 and 8E6 degrade p300 in a proteasomal-dependent, E6AP-independent manner.

(A) LXSN and E6-expressing HFKs were treated with DMSO (−) or MG132 (+) 2 hr prior to harvesting cell lysates. Lysates were then analyzed by immunoblot for p300, p53 and nucleolin (control). (B) LXSN and E6-expressing HFKs were treated with DMS0 (−) or Lactacystin (+) 2 hr prior to harvesting cell lysates. Lysates were then analyzed by immunoblot for p300, p53 and nucleolin. (C) E6AP was knocked down by transfection of LXSN or E6-expressing HFKs with a pool of 4 siRNAs targeting E6AP (+) and compared to cells mock treated by transfection with a pool of 4 non-targeting siRNAs (−). 72 hr post transfection, lysates were harvested and analyzed by immunoblot for p300, E6AP, p53 and nucleolin (control). (D) E6AP was knocked down using 2 individual siRNAs from the above pool (1 and 2) and compared to cells mock treated with 1 individual non-targeting siRNA (−). 72 hr post transfection, lysates were harvested and analyzed by immunoblot for p300, E6AP, p53 and nucleolin (control).

Figure 4. p300 degradation is modulated by AKT activity.

(A) Domain structure of p300 demonstrating the location of the AKT phosphorylation site with respect to the C/H3 and Q domains [modified from 44]. Arrows indicate the start and end of the C/EBPβ binding region that is also the binding site of many other proteins including E6 (B) Representative immunoblot showing levels of p300, CBP and nucleolin (control) in LXSN or E6-expressing HT1080s and HFKs. (C) Representative immunoblot showing levels of p300, pAKT, total AKT and Nucleolin in LXSN and E6-expressing cells. (D) LXSN and E6-expressing HFKs were treated with DMS0 (−) or Ro-31(+) 2 hr prior to harvesting cell lysates. Lysates were then analyzed by immunoblot for p300, phospho-AKT, AKT and nucleolin. (E) LXSN and E6-expressing HFKs were treated with DMS0 (−) or LY294002 (+) 24 hr prior to harvesting cell lysates. Lysates were then analyzed by immunoblot for p300, phospho-AKT, AKT and nucleolin. (F) Representative immunoblot showing levels of transiently transfected HA-p300, endogenous p300 and Actin as a loading control in LXSN, 8E6 and Δ8E6 cells transfected with WT-HAp300 or p300 S1834A/E mutants.

Figure 5. E6 and AKT compete for binding to p300.

(A) Representative immunoblot showing the levels of AKT and E6 associated with p300 under increasing molar input of E6. Input protein levels are shown at the left and represent 5% of input protein. (B) Quantitation of E6 and AKT levels from A. 20X molar GST is shown as a control. (C) Representative immunoblot showing the levels of AKT and E6 associated with p300 under increasing molar input of AKT. Input protein levels are shown at the left and represent 5% of input protein. (D) Quantitation of E6 and AKT levels from C.

Figure 6. p300 degradation alters K1, K10 and IVL expression.

(A) Representative immunoblot showing levels of K1, K10 and IVL protein in LXSN, 38E6, 8E6 and Δ8E6-expressing HFKs during 72 hr calcium-induced differentiation. Actin levels are shown as a loading control. (B) K1, K10 and IVL mRNA levels levels in LXSN 38E6, 8E6 and Δ8E6-expressing HFKs during 72 hr calcium-induced differentiation. Relative levels of each mRNA were calculated using the ΔΔCT method with GAPDH to normalize mRNA levels within each sample. Values shown are fold-change in each sample compared to the LXSN (0 hr) vector control. (C) ChIP analysis using anti-p300 antibodies to pull down chromatin from LXSN control, 8E6 and 38E6-expressing HFKs, with quantitation by real-time PCR. Values represent the enrichment of p300 at the IVL promoter shown relative to the IgG and RPL30 negative controls. Error bars represent standard deviation (n = 3, p<.01 by 2-tailed student's T-test).

Figure 7. p300 knockdown by siRNA is sufficient to attenuate the mRNA and protein expression of K1, K10 and involucrin.

(A) p300 was knocked down using either a pool of 4 siRNAs (pool) or 2 individual siRNAs (si 1 and si 2) and compared to cells mock treated with a non-targeting siRNA (CTR). 72 hr post transfection, lysates were harvested and analyzed for p300, K1, K10 and IVL mRNA levels. Relative levels of each mRNA were calculated using the ΔΔCT method with GAPDH to normalize mRNA levels within each sample. Values shown are the fold-change in each sample compared to the siRNA control. Error bars represent standard deviation (n = 2). (B) p300 was knocked down using either a pool of 4 siRNAs (pool) or 2 individual siRNAs (si 1 and si 2) and compared to cells mock treated with a non-targeting siRNA (CTR). 48 hr post transfection, cells were treated with either normal media or media containing 1.5 mM CaCl₂ to induce differentiation. 24 hr later lysates were harvested and analyzed for K1, K10 and IVL mRNA levels. Relative levels of each mRNA were calculated using the ΔΔCT method with GAPDH to normalize mRNA levels within each sample. (C) A parallel set of samples was treated as in B, and 24 hr later lysates were harvested and analyzed by immunoblot for p300, K1, K10, IVL and Actin (control).